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High Performance Liquid Chromatography - Lough W.J.

Lough W.J. High Performance Liquid Chromatography - Blackie academic, 1977. - 282 p.
Download (direct link): highperoranceliquidchromatographi1977.pdf
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A cell for an RI detector which measures the change in transmitted light intensity is shown in Figure 6.6b. As analytes flow through the cell the RI at the interface changes, and thus the transmitted intensity changes. When light strikes the interface at near-normal incidence, almost all of the light is transmitted. However, as the angle of incidence approaches the critical angle, more and more of the beam is reflected, and less passes through the interface (see Figure 6.7). The transmitted light is reflected from the scattering surface, and passes back through the prism to a photodetector. Suitable beam stops are used to reject the reflected beam. The sample and reference cells are located next to each other, separated by a gasket which also defines the distance between the prism and scattering surface. The physical proximity minimises temperature differences between the cells. More than one prism, using glass of different RI may be needed for optimum performance with a wide range of mobile phase RI, although in practice a single prism may given adequate performance for most mobile phases.
RI detectors are very useful instruments because they can be used to quantitate analytes which are otherwise difficult to detect. An example is the analysis of sugars which have poor UV absorbances and thus cannot be detected by UV absorbance or fluorescence measurements without chemical derivatisation. The exception to this universality is when the solute and solvent have identical RIs, whereupon no signal would be observed. The RI of a number of common HPLC solvents is given in Table 6.2.
6.5.2 Operation of a refractive index detector
The universal nature of the RI detector puts this device at a disadvantage in terms of sensitivity, since many factors cause changes in the RI of the
column eluent; changes in temperature and pressure are important causes of noise and drift in HPLC RI detectors. Minor pump pulsations which do not cause problems with solute property detectors may cause severe periodic baseline noise with an RI detector. The cause is easy to detect, but the cure may be more difficult. The RI detector should always be present as the last detector if more than one detector is being used. This will minimise back-pressure at the detector and thus minimise the effects of pump pulsation. Another good reason to place the RI detector last is that, with certain cell designs, severe damage to the cell will occur even at relatively low back-pressures due to a second detector connected after the RI instrument. To further reduce pump pulsation noise a pulse dampener might help, or even a change to a pulseless syringe pump if a suitable device is available. Temperature control is another important factor, since the temperature coefficient of RI is of the order of 10 3-10^Rl units/C for many solvents. Thus to be able to measure a change in RI of only 10-6 units, the temperature stability between the sample and reference cell compartments must be of the order of 0.01-0.001C. Heating or cooling of the cell caused by the effluent from the HPLC column can cause great problems; usually RI detectors are built with a lengthy piece of HPLC tubing in thermal contact with the temperature-controlled cell block to allow equilibration of the liquid temperature before it enters the cell. In practice equilibration might not be ideal, and this may lead to drift associated with flow rate changes. A further case of drift in RI detectors is change in the mobile phase composition. This can come about in a number of ways. Mobile phases which are made up of more than one component are best pre-mixed, rather than mixed using a binary pump system; this is not likely to give a sufficiently stable mobile phase composition to obtain a good baseline with an RI detector on a sensitive range. Another problem can be dissolved gas in the mobile phase causing RI changes. Either continuous helium outgassing, or no degassing at all may offer acceptable solutions. Gradient elution operation is practically impossible.
6.6 Electrochemical detectors
6.6.1 Principles of operation and design of electrochemical detectors
There are a number of electrochemical interactions which may be useful as the basis for detection in HPLC; the most commonly used electrochemical detectors are based on amperometric measurements. The principle of operation of an amperometric detector is the oxidation or reduction of analyte in a flow-through electrolysis cell with a constant applied electrical potential, e.g. the oxidation of hydroquinone,
Very low detection limits can be achieved with amperometric detectors (rivalling fluorescence detection), particularly for compounds which are oxidised or reduced at relatively low potentials. In aqueous solvents the limitation on the applied potential is the oxidation of H20 or the reduction of H + , at about -1.2 or + 1.2 V. By limiting the applied potential to that needed to achieve a good response from the target compound, selectivity against compounds with higher oxidation or reduction potentials is achieved. Figure 6.8a represents a block diagram of an amperometric detector.
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